All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
Lipids, and especially polar lipids, nitrogen containing lipids, and carbohydrate containing lipids (phospholipids, sphingosines, glycolipids, ceramides, sphingomyelins) are the major building blocks of cell membranes, tissues, etc. Additionally they play important roles in signal transduction processes and in a variety of biochemical and biosynthetic pathways.
Glycerophospholipids, lipids based on a glycerol backbone and containing a phosphate head group, are the main building blocks of cell membranes. Since most, if not all, biochemical processes involve cell membranes, the structural and physical properties of membranes in different tissues is crucial to the normal and efficient functioning of membranes in all biochemical processes.
Other important constituents of biological membranes are cholesterol, glycolipids, and peripheral and integral proteins. The basic structure of biological membranes is thus a series of recurrent unities of lipid-protein complexes. The membrane is asymmetric. The function of the external (cellular) and internal (sub cellular) membrane systems depends on their composition and on the integrity of their phospholipid structure. In addition to their presence in cell membranes, phospholipids constitute structural and functional elements of the surface mono-layers of lipoproteins and of surfactants.
Of utmost importance for the function of biological membranes is their fluidity, which is decisively influenced by phospholipids. Besides the content in cholesterol and proteins and the nature and charge of the polar head groups of phospholipids in the system, membrane fluidity depends on the length of the chains of fatty acid residues in the phospholipid molecule, as well as on the number and type of pairing of their double bonds.
Many health benefits have been attributed to the consumption of certain fatty acids. For example, it has been reported in many research studies that polyunsaturated fatty acids (PUFA) of the type omega-3 and omega-6, have several health benefits on cardiovascular disease, immune disorders and inflammation, renal disorders, allergies, diabetes, and cancer. These types of fatty acids are naturally occurring mainly in fish and algae, where they are randomly distributed on the sn-1, sn-2, and sn-3 positions of the glycerol backbone of triglycerides.
Extensive clinical studies investigating the importance of Docosahexaenoic acid (DHA; 22:6, n-3), one of the most important omega-3 fatty acids, in the brain, found that low levels of DHA are associated with depression, memory loss, dementia, and visual problems, while a dramatic improvement in the elderly brain function has been observed as blood levels of DHA increase.
Fatty acid differences, including DHA have been shown in the brain of Alzheimer's patients as compared with normal age-matched individuals. Furthermore, low serum DHA is a significant risk factor for the development of Alzheimer's patients. Recently, it was shown [Conquer et al. (2000) Lipids; 35(12):1305-1312] in addition to Alzheimer's patients that in other dementias and cognitively impaired but non-demented individuals, there are low levels of n-3 fatty acids in the plasma. Suggesting that the decreased level of plasma DHA was not limited to the Alzheimer's disease patients but appears to be common in cognitive impairment with aging, and therefore may be a risk factor for cognitive impairment and/or dementia.
Other known benefits of DHA include: lower risk of arrhythmias, reduction in the risk of sudden cardiac death, lower plasma triglyceride levels and reduced blood clotting tendency. Furthermore, DHA may have importance in the field of brain functioning enhancement, baby formula fortification, diabetics and cancer.
The human body does not adequately synthesize DHA. Therefore it is necessary to obtain it from the diet. Humans obtain DHA from their diets, initially through the placenta, then from breast milk (or baby formula), and later through dietary sources, such as fish, red meats, animal organ meats and eggs. Popular fish like tuna, salmon and sardines are rich sources. Until recently, the primary source of DHA dietary supplements has been fish oils. The ability of enzymes to produce the omega-6 and omega-3 family of products of linoleic and alpha-linolenic acid, declines with age. Thus, because DHA synthesis declines with age, as people get older their need to acquire DHA directly from diet or supplements increases. In fact, several recent publications suggested DHA to be considered as an essential fatty acid [e.g. Muskiet, F. et al. (2004) J Nutr. 134(1):183-6].
Because DHA is important for signal transmission in the brain, eye and nervous system, many consumers concerned with maintaining mental acuity are searching for a pure, safe way to supplement their DHA levels.
Polyunsaturated acids, in particular long chain, Such as omega-3 and 6, have been shown to confer many valuable health benefits on the population. The global market for long-chain PUFAs, including the food segment, is rapidly growing.
The majority of efforts in the industry are however invested in the improvement of PUFA processing techniques and in the creation of higher concentrated grades of PUFA derivatives to accommodate dietary supplements and functional foods needs.
PUFA-Lipids
PS-PUFA (Serine Glycerophospholipid—PUFA Conjugates)
Phosphatidylserine (PS) is the major acidic phospholipid in the brain, being one of the most important building blocks of cerebral cell membranes. The level of PS in brain cell membranes ensures their fluidity and functional structure, while guaranteeing normal and efficient signal transduction processes, efficient glucose consumption, and other biological pathways that result in normal cognitive and mental functions.
PS is a natural phospholipid with bio-functionality that has made it one of the most promising dietary supplements in the field of brain nutrition, for its properties in a variety of cognitive and mental functions. PS has been shown to improve memory, slow cognitive decline, especially in the elderly, fight dementia and early stages of Alzheimer's disease, reduce stress and tension, improve attention span, enhance mood and fight depression, to name but few.
PS is not abundant in human nutrition. Moreover, the biosynthetic pathways responsible for the production of PS are malfunctioning in many people, especially the elderly, resulting in low levels of PS in the body and brain, which results in a variety of cognitive and mental disorders, such as depression, memory loss, short attention span, learning difficulties, etc. The supplementation of PS in the diets of elderly people with such disorders has resulted in dramatic improvements of these disorders. Over the recent years, studies have shown that even younger people can benefit from dietary supplementation of PS. PS has been shown to improve the learning capabilities of students, improve memory and attention span, etc.
Interestingly, early attempts to elucidate the role of DHA in rat developing brain had demonstrated that intra-amniotic injection of DHA to E17 fetal rats resulted with redistribution of total brain PL, and specifically 56.4%, increase in PS-DHA abundance [Green et al. (1995) J. Neurochem; 65(6):2555-25560].
It is therefore an object of the present invention to provide special conjugated preparations of PS, for use mainly as nutraceuticals, pharmaceuticals, medical foods and as functional food additives.
Studies conducted with PUFA-containing phospholipids (conjugated glycerophospholipids) have shown the following:    1. They are high-energy, basic, structural, and functional elements of all biological membranes, such as cells, blood corpuscles, lipoproteins, and the surfactant.    2. They are indispensable for cellular differentiation, proliferation, and regeneration.    3. They maintain and promote the biological activity of many membrane-bound proteins and receptors.    4. They play a decisive role for the activity and activation of numerous membrane-located enzymes, such as sodium-potassium-ATPase, adenylyl cyclase and lipoprotein lipase.    5. They are important for the transport of molecules through membranes.    6. They control membrane-dependent metabolic processes between the intracellular and intercellular space.    7. The polyunsaturated fatty acids contained in them, such as linoleic and linolenic acid, are precursors of the cytoprotective prostaglandins and other eicosanoids.    8. As choline and fatty acid donors they have an influence in certain neurological processes.    9. They emulsify fat in the gastrointestinal tract.    10. They are important emulsifiers in the bile.    11. They codetermine erythrocyte and platelet aggregation.    12. They influence immunological reactions on the cellular level.
Phospholipids containing PUFA are theoretically of importance in all those diseases in which damaged membrane structures, reduced phospholipid levels, and/or decreased membrane fluidity are present. This hypothesis is supported by experimental and clinical investigations of various membrane-associated disorders and illnesses.
Studies on the active principle as well as pharmacological and clinical trials are available on a variety of conditions and diseases related to membrane damage. For example in various liver diseases, hepatocyte structures are damaged by, for example, viruses, organic solvents, alcohol, medicaments, drugs, or fatty food. In consequence, membrane fluidity and permeability may be disturbed, and membrane-dependent metabolic processes as well as membrane-associated enzyme activities may be impaired, considerably inhibits liver metabolism.
Other examples include hyperlipoproteinemia with or without atherosclerosis, hemorrheological disturbances with an elevated cholesterol/phospholipid ratio in the membranes of platelets and red blood cells, neurological diseases, gastrointestinal inflammations, kidney diseases, and in a variety of aging symptoms.
All these very different diseases have in common comparable membrane disorders. With polyunsaturated phosphatidylcholine molecules such disorders may be positively influenced, eliminated, or even improved beyond normal due to the high content in polyunsaturated fatty acids. Following are some examples of the mechanisms that mediate this phenomenon:    1. High-density lipoprotein (HDL) particles enriched with PUFA-containing-phosphatidylcholine are able to take up more cholesterol from low-density lipoprotein (LDL) and tissues. More cholesterol can be transported back to the liver. This action on the cholesterol reverse transport is unique. All other lipid-lowering agents reduce either the cholesterol absorption in the body or the cholesterol synthesis in the liver and its distribution to the periphery. These substances, however, do not physiologically mobilize the cholesterol already present in the periphery.    2. The cholesterol/phospholipid ratio in membranes, platelets, and red blood cells decreases and membrane function is improved up to normalization.    3. Peroxidative reactions are reduced, damaged hepatocyte membrane structures restored, membrane fluidity and function stabilized, immuno-modulation and cell protection improved, and membrane-associated liver functions enhanced.    4. With the normalization of the cholesterol/phospholipid ratio, the bile is also stabilized.    5. Due to its specific property as a surface-active emulsifier, PUFA-containing-phosphatidylcholine solubilize fat and is used in reducing the risk and treatment of fat embolism.    6. The substitution with poly-unsaturated-fatty-acids and choline may have a cytoprotective effect in the brain and activate neuronal processes.    7. Liposomes with polyunsaturated phosphatidylcholine molecules may act as drug carriers, such as of vitamin E.
Some publications have reported preparations of phospholipids and suggested their use in neurological or psychiatric conditions.
WO 97/39759 discloses preparations comprising dieicosapentanoylphosphatidylcholine, didocosahexaenoylphosphatidylcholine, 1-eicosapenta-enoyl, 2-docosahexaenoylphosphatidylcholine, and 1-docosahexaenoyl, 2-eicosa-pentaenoylphosphatidylcholine, which are useful for treating bipolar disorders.
JP 06256179 discloses preparations comprising 1,2-diacyl-sn-glycerol derivatives of formula R1-0-CH2—CH(OR2)—CH2-0-R3 (I) wherein R1=14-24C saturated or monoene fatty acid residue; R2=a residue of arachidonic acid, eicosapentaenoic acid (EPA) or DHA; R3=H, phosphorylcholine, phosphoryl-ethanolamine, phosphorylserine or phosphorylinositol, 1-Oleoyl-2-docosahexaenoyl-sn-glycero-3-phosphorylcholine that are effective components for improving learning ability and for treating senile dementia. However, none of the preparations disclosed in JP 06256179 comprises DHA.
JP 06279311 discloses phosphatidylserine derivatives of formula (I) and their salts wherein R1=acyl residue of myristic, palmitic or stearic acid; R2=acyl residue of linoleic, linolenic, arachidonic or docosahexaenoic acid, and their use in the treatment of senile dementia accompanied with central nervous lesions, especially Alzheimer's disease. However said compositions do not comprise EPA as a possible substituent on the glycerophospholipid backbone.
The utilization of phospholipids enriched with PUFA holds many potential advantages from a clinical point of view. The phospholipid may deliver the essential fatty acid to specific organs or body parts, such as the brain, and assist in the incorporation of these fatty acids in membranes. Other advantages may arise from the fact that phospholipids enriched with PUFA will not have odor problems such as found in the major current nutraceutical source, the fish oils. Furthermore, some preliminary clinical studies have shown that PUFA incorporated in phospliolipids possess superior efficacy than PUFA carried by triglycerides. [Song et al. (2001) Atherosclerosis, 155, 9-18].
Further studies have shown that the activity of DHA-rich phospholipids was different from that of DHA-rich triacylglycerols in spontaneously hypertensive rats [Irukayama-Tomobe et al. (2001) Journal of Oleo Science, 50(12), 945-950]. Spontaneously hypersensitive rats (SHR) were fed test lipid diets for six weeks, which contained 30%-DHA phospholipid (DHA-PL) extracted from fish roe or 30%-DHA fish oil (DHA-TG). The control diet contained corn oil in the presence of test lipids. After feeding, blood pressure in the DHA-TG and DHA-PL diet groups was found significantly lower compared to the control. Serum fatty acid content of dihomo-linoleic acid (DHLnA) and Arachidonic acid (ARA; 20:4n-6) of the DHA-PL diet group was significantly less than the control or DHA-TG diet group. Serum triacylglycerol, phospholipid and total cholesterol in the DHA-TG and DHA-PL diet groups were significantly less than in the control. Liver total cholesterol in DHA-PL was twice that in the DHA-TG diet group and control.
Many PUFA-containing agents suffer from stability and quality problems due to the high degree of oxidation of the polyunsaturated fatty acids. These problems require the incorporation of antioxidants as well as the utilization of special measures which attempts to reduce this oxidation. The utilization of phospholipids as carriers of PUFA may result in enhanced stability of such products due to the anti-oxidative properties of phospholipids.
It seems that one of the most effective transport mechanisms for such essential fatty acids is the attachment of these groups to phospholipid molecules. The phospholipids have been shown to pass through the blood-brain barrier and transport the DHA where it is needed.
Linoleic acid (LA, C18:2, ω-6) and α-linolenic acid (ALA, C18:3, ω-3), are classified as essential fatty acids (EFA). The body cannot synthesize them de novo, and they must therefore be obtained through food sources providing them “ready-made”. Both LA and ALA are needed for optimal growth and good health. Both LA and ALA are precursors of the ω-3 and ω-6 PUFA. LA is required for the synthesis of arachidonic acid (AA), a key intermediate in the synthesis of eicosanoids, whereas ALA is used partly as a source of energy, and partly as a precursor for metabolites and longer chain PUFA. Within the human body LA and ALA can be elongated and desaturated to other more unsaturated fatty acids, principally arachidonic acid (C20:4, ω-6) and DHA (C22:6, ω-3).
Soybeans, egg yolk, bovine brain and fish are the major natural sources for obtaining and producing phospholipids, especially PS. The type of fatty acyl residues at the sn-1 and sn-2 positions in natural phospholipids vary, and their proportion in general depends on their source. For example, soybean is rich with LA fatty acid (about 54%) whereas fish derived lecithin is abundant with DHA fatty acid residue. The PS extracted from animal brain tissues, similar to human brain PS, has a fatty acid composition which is characterized by relatively high levels of omega-3 moieties, compared to the levels of omega-3 found in plants, such as soy phospholipids. The bio-functionality of soybean PS in the improvement of cognitive function has been shown to be different from that of human brain PS [WO 2005/037848].
Organoleptic Concerns
PUFAs are traditionally extracted from coldwater fish. Despite the healthy image, one of the problems of consumer acceptance has been the resulting strong, fishy taste. To address this, microencapsulated forms of omega-3 have been pioneered in the last 15 years. A further step was the development of egg-containing products such as DHA-enriched mayonnaise and pasta. DHA-enriched yoghurts, baked goods and broilers were also envisaged.
There is no other nutritional product or preparation that is considered to be an agent of PUFA delivery. All current commercial products are based on the fatty acids themselves in an encapsulated form or on foods enriched with PUFA through special animal/crop feed.
ADHD
Attention-deficit/hyperactivity disorder (ADHD) encompasses a broad constellation of behavioural and learning problems and its definition and diagnosis remain controversial [Kamper (2001) J. Pediatr. 139:173-4; Richardson et al. (2000) Prostaglandins Leukot. Essent. Fatty Acids, 63(1-2):79-87]. The etiology of ADHD is acknowledged to be both complex and multi-factorial. Traditionally, ADHD is the diagnosis used to describe children who are inattentive, impulsive, and/or hyperactive. A conservative estimate is that 3-5% of the school-age population has ADHD [American Psychiatric Association (1994) Diagnostic and statistical manual of mental disorders. 4th ed. (DSM-IV) Washington, D.C.]. Roughly 20-25% of children with ADHD show one or more specific learning disabilities in math, reading, or spelling [Barkley, R. A. (1990) Attention-deficit hyperactivity disorder: a handbook for diagnosis and treatment. New York: Guilford Press]. Children with ADHD often have trouble performing academically and paying attention, and may be disorganized, have poor self-discipline, and have low self-esteem. Treatments for ADHD include behavior therapy and medications, mainly methylphenidate (Ritalin™). Psychostimulant drugs and antidepressants are often used to calm children with ADHD, with an effectiveness rate of ˜75% [Swanson et al. (1993) Except Child 60:154-61]. The advantages of Using these medications include rapid response, ease of use, effectiveness, and relative safety. Disadvantages include possible side effects, including decreased appetite and growth, insomnia, increased irritability, and rebound hyperactivity when the drug wears off [Ahmann et al. (1993) Pediatrics; 91:1101-6]. Moreover, these medications do not address the underlying causes of ADHD. Thus, studies to elucidate the potential contributors to the behavior problems in ADHD may lead to more effective treatment strategies for some children.
Omega-3 fatty acids are specifically implicated in maintaining central nervous system function. Deficiency of n-3 fatty acids in rats and monkeys has been associated with behavioral, sensory, and neurological dysfunction [Yehuda et al. (1993) Proc. Natl. Acad. Sci. USA; 90:10345-9; Reisbick et al. (1994) Physiol. Behav. 55:231-9; Enslen et al. (1991) Lipids; 26:203-8]. Several studies have focused on essential fatty acid metabolism in children with ADHD [Colquhloun et al. (1981) Med Hypotheses; 7:673-679]. Children with hyperactivity have been reported to be thirstier than normal children and have symptoms of eczema, asthma, and other allergies [Mitchell et al. (1987) Clin. Pediatr.; 26:406-11]. For example, in a cross-sectional study in 6 to 12-year-old boys recruited from central Indiana, it was showed that 53 subjects with ADHD had significantly lower proportions of key fatty acids in the plasma polar lipids ARA, eicosapentaenoic acid (EPA; 20:5n-3), and DHA and in red blood cell total lipids (20:4n-6 and 22:4n-6) than did 43 control subjects [Stevens et al. (1995) Am. J. Clin. Nutr.; 62:761-8]. However, recent publications investigating whether DHA supplementation would result in amelioration of the symptoms in ADHD children, suggested that careful attention should be paid as to which fatty acid(s) is used [Hirayama et al. (2004) Eur. J. Clin. Nutr.; 58(3):467-73; Voigt et al. (2001) J Pediatr.; 139(2):189-96]. In these studies DHA supplementation had demonstrated only marginal if any beneficial effects.
Recently, it has been suggested that one of the possible solutions to the nutrient deficiencies which are common in ADHD, could be PS supplementation [Kidd (2000) Alter Med Rev.; 5(5):402-28].
It is therefore an object of the present invention to provide lipid preparations enriched with omega-3 or omega-6 fatty acids, for use mainly as nutraceuticals and as functional food additives. The composition of said preparation is such that it provides the preparation with the property of enhancing the bioavailability of PUFAs. Thus upon its consumption, preferably in the form of nutraceuticals, food additives or pharmaceutical compositions, the organism may, in the most efficient way, enjoy the benefits provided by said preparation, as will be described in detail below.
This and other objects of the invention will become apparent as the description proceeds.